Targeting of multiple tumor-associated antigens by individual T cell receptors during successful cancer immunotherapy.

The T cells of the immune system can target tumors and clear solid cancers following tumor-infiltrating lymphocyte (TIL) therapy. We used combinatorial peptide libraries and a proteomic database to reveal the antigen specificities of persistent cancer-specific T cell receptors (TCRs) following successful TIL therapy for stage IV malignant melanoma. Remarkably, individual TCRs could target multiple different tumor types via the HLA A∗02:01-restricted epitopes EAAGIGILTV, LLLGIGILVL, and NLSALGIFST from Melan A, BST2, and IMP2, respectively. Atomic structures of a TCR bound to all three antigens revealed the importance of the shared x-x-x-A/G-I/L-G-I-x-x-x recognition motif. Multi-epitope targeting allows individual T cells to attack cancer in several ways simultaneously. Such "multipronged" T cells exhibited superior recognition of cancer cells compared with conventional T cell recognition of individual epitopes, making them attractive candidates for the development of future immunotherapies.

Emergence of immune escape at dominant SARS-CoV-2 killer T cell epitope.

We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as "variants of concern," was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants.

Resistant silkworm strain block viral infection independent of melanization.

Melanization mediated by the prophenoloxidase-activating system (proPO) is an important immune response in invertebrates. However, the role of melanization on viral infection has not been wildly revealed in Bombyx mori (B. mori), silkworm. Here, we investigated the extent of melanization of susceptible (871) and resistant (near-isogenic line 871C) B. mori strains. The result showed that the extent of melanization was significantly higher in the susceptible strain than in the resistant strain. A majority of Serpins were up-regulated in the resistant strain through iTRAQ-based quantitative proteomics, comparing with susceptible strain. Our data further identified that Serpin-5, Serpin-9 and Serpin-19 reduced PO activity, indicating that the menlanization pathway was inhibited in the resistant strain. Moreover, our results indicated that the hemolymph of 871 reduced viral infection in the presence of PTU, indicating that melanization of 871 strain hemolymph blocked viral infection. However, viral infection was significantly suppressed in the hemolymph of 871C strain regardless of the presence of PTU or not, which implied that the resistant strain inhibited viral infection independent of the melanization pathway. Taken together, our findings indicated that the melanization pathway was inhibited in resistant strain. These results expend the analysis of melanization pathway in insects and provide insights into understanding the antiviral mechanism.

Accumulation and toxicological response of atrazine in rice crops.

Atrazine is one of the most widely used herbicides for controlling weeds and grasses. Due to its intensive use, it has become a serious contaminant in soil and water. To evaluate impact of atrazine on graminaceous crops, experiments focusing on atrazine accumulation and toxic response in rice (Oryza sativa) were carried out. Treatment with atrazine at 0.05-0.8 mg L(-1) for 6 d reduced elongation of shoot and root. Compared with a mock treatment, the elongation of shoot with atrazine was 67.1 percent of the control, whereas that of root was 79.5 percent, indicating that the shoot was more affected than the root. Atrazine was readily absorbed by rice from media. Although the quantitative absorption of atrazine was positively correlated with the external supply of the herbicide, translocation of atrazine from roots to the above-ground was reduced from 39.88±6.26 (at 0.05 mg L(-1)) to 9.25±0.27 (0.8 mg L(-1)). While accumulation of atrazine in rice plants led to toxic responses such as over-generation of hydrogen peroxide and superoxide anions, it triggered the plant defense system against the herbicide-induced oxidative stress. This was best presented by the enhanced activities of several antioxidant enzymes (e.g. superoxide dismutase, catalase and peroxidase) and expression of genes responsible for the tolerance to atrazine toxicity.

Scutellarin regulates osteoarthritis in vitro by inhibiting the PI3K/AKT/mTOR signaling pathway.

Osteoarthritis (OA) is a highly prevalent disease worldwide that causes disability and diminishes the quality of life of affected individuals. The disease is characterized by cartilage destruction, increased inflammatory responses and cholesterol metabolic disorder. Scutellarin is the major active ingredient extracted from Erigeron breviscapus, and it has been demonstrated to possess various pharmacological functions in the treatment of the disease. However, its effects on OA are complex. The present study investigated whether scutellarin can mediate the release of inflammatory cytokines, the expression of collagen- and cholesterol-related proteins, and regulate the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in a cell model of OA. Interleukin (IL)-1ß was used to stimulate OA in SW1353 cells in vitro. The primary methods used were ELISA and western blotting, which were carried out to examine the effects of scutellarin on the cell model of OA. It was found that scutellarin increased the expression of collagen II and SRY-box 9, whereas it suppressed the expression of matrix metalloproteinase 13. In addition, scutellarin downregulated the expression levels of cholesterol 25-hydroxylase and cytochrome P450 family 7 subfamily B polypeptide 1, but upregulated the expression of apolipoprotein A-1 and adenosine triphosphate-binding cassette transporter A1. The IL-1ß-induced increase in the expression of IL-6 was decreased by treatment with scutellarin; however, scutellarin did not alter the expression of C-reactive protein and tumor necrosis factor-α. The protein expression levels of AKT, phosphorylated (p)-AKT, mTOR and p-mTOR in the PI3K/AKT/mTOR signaling pathway were decreased in the IL-1ß-induced SW1353 cells following scutellarin treatment. Overall, the findings of the present study demonstrated that scutellarin regulated OA in vitro by inhibiting the PI3K/AKT/mTOR signaling pathway.

C-lysozyme contributes to antiviral immunity in Bombyx mori against nucleopolyhedrovirus infection.

Lysozymes is a ubiquitous immune effector that is widely distributed in both vertebrates and invertebrates. Previous reports have shown that lysozymes significantly inhibit viral infections in vertebrates. However, the antiviral effects of lysozymes in invertebrates remain unclear. Here, we investigated the role of lysozymes in Bombyx mori (B. mori) response to viral infection by overexpressing B. mori C-lysozyme (BmC-LZM) in larvae and cells. We found that BmC-LZM was up-regulated in cells in response to viral infection. Indeed, the overexpressing of BmC-LZM significantly inhibited viral replication in cells during late-stage infection. However, this effect was reversed by BmC-LZM mRNA. BmC-LZM was successfully overexpressed in B. mori strain 871 using Baculovirus Expression Vector System (BEVS). This overexpression markedly reduced viral proliferation and increased larval survival percentage. Thus, BmC-LZM inhibited viral replication both in vivo and in vitro, indicating that BmC-LZM is involved in the insect immune response to viral infection. Our results provide a basis for further applications of lysozymes.

A collection of glycosyltransferases from rice (Oryza sativa) exposed to atrazine.

The rice (Oryza sativa) GTs belong to a super family possibly with hundreds of members. However, which GTs are involved in plant response to toxic chemicals is unknown. Here, we demonstrated 59 novel GT genes screened from our recent genome-wide sequencing datasets of rice crops exposed to atrazine (a herbicide persistent in ecosystems). Analysis of GT genes showed that most of the GTs contain functional domains typically found in proteins transferring glycosyl moieties to their target compounds. A phylogenetic analysis revealed that many GT genes from different families have diverse cis-elements necessary for response to biotic and environmental stresses. Experimental validation for the GTs was undertaken through a microarray, and 36 GT genes were significantly detected with an expression pattern similar to that from deep-sequencing datasets. Furthermore, 12 GT genes were randomly selected and confirmed by quantitative real-time RT-PCR. Finally, the special activity of total GTs was determined in rice roots and shoots, with an increased activity under the atrazine exposure. This response was closely associated with atrazine absorption in the rice tissues. These results indicate that exposure to atrazine can trigger specific GT genes and enzyme activities in rice.

Inactivation of jack bean urease by scutellarin: elucidation of inhibitory efficacy, kinetics and mechanism.

In the present study, the inactivation effect of scutellarin (SL) on jack bean urease was investigated to elucidate the inhibitory potency, kinetics and mechanism of inhibition. It was revealed that SL acted as a concentration- and time-dependent inactivator of urease characteristic of slow-binding inhibition with an IC50 of 1.35±0.15 mM. The rapid formation of the initial SL-urease complex with an inhibition constant of Ki=5.37×10(-2) mM was followed by a slow isomerization into the final complex with the overall inhibition constant of Ki*=3.49×10(-3) mM. High effectiveness of thiol protectors, such as L-cysteine (L-cys), 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) significantly slowed down the rate of inactivation, indicating the strategic role of the active site sulfhydryl group in the blocking process. While the insignificant protection by boric acid and fluoride from the inactivation further confirmed that the active site cysteine should be obligatory for urease inhibition, which was also rationalized by the molecular docking study. The inhibition of SL on urease proved to be reversible since SL-blocked urease could be reactivated by DTT application and multidilution. The results obtained indicated that urease inactivation resulted from the reaction between SL and the sulfhydryl group.

Experimental study on anti-inflammatory activity of a TCM recipe consisting of the supercritical fluid CO2 extract of Chrysanthemum indicum, Patchouli Oil and Zedoary Turmeric Oil in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Chrysanthemum indicum (Compositae) Linné, Pogostemon cablin (Blanco) Benth and Curcuma wenyujin (Zingiberaceae) Y. H. Chen et C. Ling are three of the extensively used herbal remedies among traditional Chinese medicines for the purpose of anti-inflammation. A traditional Chinese medicine (TCM) recipe named CPZ consisting extracts of the above three herbs, has shown noteworthy anti-influenza activity, which is closely related to its anti-inflammatory feature. AIM OF THIS STUDY: To investigated the anti-inflammtory activity of CPZ in vivo for a further exploration of the recipe's anti-inflammatory properties. MATERIALS AND METHODS: The anti-inflammatory property of CPZ on acute inflammation was evaluated by inflammatory models of dimethylbenzene (DMB)-induced ear vasodilatation and acetic acid-induced capillary permeability enhancement in mice, as well as the carrageenan-induced paw edema rat model, in which inflammation-related cytokine including prostaglandin E(2) (PGE(2)), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) in the edematous paw tissue were determined by enzyme linked immunosorbent assay (ELISA). Moreover, effect of CPZ on chronic inflammation was observed through granuloma formation in rats subjected to cotton pellet implantation. RESULTS: CPZ (340, 170, and 85 mg/kg for mice, p.o.) not only decreased the DMB-induced ear vasodilatation but also attenuated capillary permeability under acetic acid challenge in mice. And the significant inhibition on carrageenan-induced paw edema was observed. Further more, the ELISA results showed that CPZ (170, 85, and 42.5 mg/kg for rats, p.o.) could up-regulate the level of IL-1ß in the edema paw tissue of rats significantly while down-regulate that of PGE(2), but no apparent effect on TNF-α or NO was observed in the test. Besides, CPZ had a certain degree of restraining effect on the cotton pellet-induced granuloma formation in rats and the highest dose of 170 mg/kg even showed a significant suppression on it. CONCLUSION: The above results indicated that CPZ possessed a potent anti-inflammatory activity, which is indicated to be closely associated with its regulation on IL-1ß and PGE(2) thereby mediating the inflammatory response acting at an appropriate level.

Anti-Candida albicans activity and pharmacokinetics of pogostone isolated from Pogostemonis Herba.

The present work was designed to evaluate the in vitro and in vivo anti-Candida activity of pogostone (PO), a natural product isolated from Pogostemon cablin (Blanco) Benth. PO showed potent in vitro activity against clinical Candida spp. isolates tested in this study. PO and the reference drug voriconazole (VRC) were equally effective against all the fluconazole-resistant Candida albicans strains, with MIC ranging from 3.1 µg/ml to 50 µg/ml. Besides, PO was fungicidal against all Candida isolates with MFC ranging from 50 µg/ml to 400 µg/ml. By contrast, VRC was fungistatic as it failed to elicit a fungicidal effect against the Candida spp. isolates at the highest tested concentration (400 µg/ml). Furthermore, oral and topical PO administration effectively reduced the fungal load in vagina of vulvovaginal candidiasis mouse models. Topical PO administration (1.0-4.0 mg/kg) demonstrated higher activity against the vulvovaginal candidiasis than VRC (4.0 mg/kg). The pharmacokinetics and safety profile of PO were also investigated. The pharmacokinetics assay revealed that PO was easily absorbed after oral administration in mice, which might account for its in vivo anti-Candida effect. The acute toxicity test showed that the median lethal dose of PO in mice was 355 mg/kg, which was much higher than the daily dose used for the therapeutic experiments. This study demonstrated the potential of PO as a promising candidate for the treatment of Candida infections, particularly for vulvovaginal candidiasis.